Liquids, mixtures, solutions and reacting mixtures are often characterized using optical techniques such as photometry, spectrophotometry, fluorometry, or spectrofluorometry. In order to characterize samples of these liquids, conventional methods and apparatus generally employ a sample-holding vessel or cell, for instance, a cuvette, which has two or more sides of optical quality so as to permit the passage of those wavelengths needed to characterize a liquid contained therein.
Unfortunately, biological sampling techniques often yield very small quantities of material for analysis. Accordingly, absorbance and fluorescence measurements with minimal consumption of sample material have become paramount. When dealing with very small sample volumes—for instance, from 1 to 2 microliters—it is difficult to create cells or cuvettes small enough to be filled and permit the industry standard 1 cm optical path to be used. It is also difficult and/or time consuming to clean these cells or cuvettes for use with another sample. Thus, conventional methods of photometry, spectrophotometry, fluorometry, spectrofluorometry, etc. are impractical when dealing with small sample volumes, such as of biological samples produced by laser-capture microdissection.
In the case of photometry or spectrophotometry, the usual quantity of interest is absorbance, A, which, for liquid samples, is most often defined as:A=−log10(T)=−log10(IR/I0)  Eq. 1;where T is the transmittance, IR the intensity (e.g., power) of light transmitted through the sample being measured and I0 is the intensity of light transmitted through a blank or reference sample. Most commonly, the absorbance value is measured in a cell or cuvette with a 1 cm path length. However, Lambert's Law states that for a collimated (all rays approximately parallel) beam of light passing through a homogeneous solution of uniform concentration the absorbance, A, is proportional to the path length through the solution. For two light path lengths P1 and P2,
                                                        A              1                                      A              2                                =                                    P              1                                      P              2                                      ;                            Eq        .                                  ⁢        2            where A1 and A2 are the absorbance values determined at path lengths P1 and P2, respectively. Further, absorbance is a function of absorptivity, c, path length, P, and analyte concentration, c, through the relation:A=εcP  Eq. 3.
Thus, it is often possible to measure absorbance using path lengths other than 1 cm and to use the results to calculate concentration or absorptivity or, if desired, to correct absorbance to the equivalent value for a 1 cm path for more-ready comparison with conventional data.
U.S. Pat. Nos. 6,809,826 and 6,628,382, each of which is incorporated herein by reference in its entirety, teach methods and apparatus of spectrophotometry or the like on extremely small liquid samples. The sample path lengths in the range of 0.2 to 2 mm taught in the above-referenced patents can be used to generate absorbance values that can be easily corrected to the 1 cm path equivalent.
According to the teachings in the above referenced patents, a sample droplet is held between two opposing substantially parallel surfaces by interfacial tension and one surface is controllably moved toward and away from the other. To provide and transmit light through the droplet for measurement, and to collect light for measurement, at least one of the surfaces may have a portion of optical measurement quality. This may be accomplished by providing at least a portion of at least one of the surfaces as a polished end of an optical fiber, wherein each such optical fiber may be finished flush with the surrounding surface portion. Typically, such surrounding surface portion often includes the surface of an end of a standard fiber optic connector or other fiber holder.
As disclosed in the above-noted patents, to make a measurement of less than about 2 micro-liters of a sample, such an amount is pipetted directly onto one of the surfaces, for instance the lower surface 15 shown in FIGS. 1A and 2A. An upper surface, surface 13, subsequently moves down so as to engage the sample and then moves upward and away from the lower surface, thus using interfacial tension to adhere to lower surface 15 and upper surface 13, wherein surface tension forms a liquid column 14 of mechanically controlled path length (see FIGS. 1A-1B and FIGS. 2A-2B). The shape and nature of the upper and lower surfaces, i.e., surface 13 and surface 15, serve to maintain the sample within a pre-defined optical path. One of the surfaces, e.g., surface 13, can be swung clear of the other for easy cleaning between measurements on different samples.
Moreover, a differential absorbance path can be employed, as shown in FIGS. 1A and 1B as well as in FIGS. 2A-2B. By measuring transmitted light intensity I at each of one or more path lengths, the difference in transmitted intensity can be used in conjunction with the known path length difference to calculate the sample absorbance. Measurements are taken as shown in FIG. 1A, where sample 14 is shown with a relatively long light path length P1 through the sample and as in FIG. 1B where sample 14 is shown with a relatively short light path length P2 through the sample. These path lengths are measured between two surfaces mutually facing one another, as discussed above, e.g., between surface 13 of an upper member 12 and surface 15 of a lower member 16. During measurements, light is delivered into the sample through one of the two surfaces and the proportion of the light transmitted through the sample is collected from the sample through the other one of the surfaces. The upper and lower members may be referred to as upper and lower anvils or pedestals, respectively. However, while anvils or pedestals are beneficial configurations, it is to be noted that such terminology does not express or imply any particular geometric form for the upper and lower members.
The difference in light path length ΔP (=═P2−P1═) may be used to calculate the optical absorbance of the sample 14 shown in FIGS. 1A-1B and FIGS. 2A-2B, since ΔP may frequently be known with a greater degree of accuracy and precision than either of P1 and P2. The path length itself may be controlled by a movement means, such as, for example, by a solenoid mounted below the apparatus, the plunger of which can bear on a pin of a hinged swing arm holding the upper member. The up and/or down movement of the plunger causes the swing arm to rotate slightly about its hinge, thus causing the upper member, displaced from the pin, to move up and/or down so as to vary the light path length through the sample.
FIGS. 2A and 2B specifically shows an additional arrangement of the previously described apparatus in which the upper and lower members include optical fiber connectors or holders and in which a first optical fiber 18a passes through the first member and a second optical fiber 18b passes through the second member. Light is delivered into the sample from one of the two optical fibers and the proportion of the light transmitted through the sample is collected from the sample by the other one of the optical fibers.
Accordingly, the configurations shown in the figures described above enable differences in transmitted intensity to be used in conjunction with known differences in path length through a desired sample in order to calculate the sample's absorbance at one or more wavelengths of interest.
When sample absorbance, A, is high, transmission, T, through the sample is low, and vice versa. One frequently desires to have a sufficiently concentrated sample or a sufficiently long path length in order to provide an absorbance of sufficient magnitude to be measurable. If the absorbance is too low, then so-called “shot” noise from the relatively high level of transmitted light may interfere with the measurement. On the other hand, providing a sample with too great of an absorbance can cause the level of measured transmitted light to be too low, whereby electronic or other system background noise can preclude or obscure accurate determination of absorbance value. Such competing effects suggest that there will be an optimal level of absorbance at which the signal-to-noise of absorbance can be maximized.
Accordingly, there is a need to provide instruments that can rapidly vary the absorbance of a sample by varying the light path length so as to assure absorbance measurements with optimal signal-to-noise characteristics. Moreover, to also assure optimal signal-to-noise characteristics, there is an additional need for precisely controlling the position of the respective optical elements, such as, for example, a pair of optical fibers so as to not only minimize circular error resulting from the instrument but also for precise measurement of variable path lengths (e.g., P1 and P2) obtained for a sample while held in constrained surface-tension mode positions so as to accurately calculate ΔP and thus accurately provide for obtained absorbance and other related instrument measurements. The present invention is directed to such a need.